Consistent production of cost-effective LongSAGE libraries

Allison C. Crawford
Jessica F. White, Southern Cross University
Peter C. Bundock, Southern Cross University
Giovanni Cordeiro, Southern Cross University
Shane R. McIntosh, Southern Cross University
Toni Pacey-Miller, Southern Cross University
Lee Rooke, Southern Cross University
Robert J. Henry, Southern Cross University

Abstract

Serial analysis of gene expression (SAGE) and related techniques are gaining popularity as tools for exploring expression of plant genes but remain suboptimal because of smaller-than-expected average concatemer sizes. The presence of low-molecular-weight contaminants in high-molecular-weight concatemer fractions reduces the average size of cloned fragments, thereby limiting the viability of high-throughput sequencing methods. Implementation of an additional digestion step to promote formation of linear concatemer fragments appears to reduce the proportion of contaminants indirectly, but with variable results. We explored the effect of initial ditag polymerase chain reaction (PCR) quantity on the average size of cloned concatemers from the greater than 1000-bp fraction. The quantity of PCR material used was found to have a strong influence on the frequency of low-molecular-weight contaminants within this fraction, which has important implications for reducing costs associated with high-throughput sequencing of concatemer clones.